PROJECT SUMMARY Quorum sensing is a form of cell-cell communication that allows members of a population to coordinate activities in a cell density-dependent fashion. Quorum sensing has been shown to play a significant role in the virulence of Pseudomonas aeruginosa and other pathogens. This research program is focused on P. aeruginosa, which controls a battery of virulence factors by acyl-homoserine lactone quorum sensing. We have been, and will continue to be, interested in basic mechanisms of quorum sensing, the selective pressures favoring quorum-sensing control of gene expression, and the costs and benefits of quorum sensing in P. aeruginosa. Quorum sensing functions to control and coordinate cooperative behaviors. It is clear that cooperativity is an evolved biological phenomenon, but there is considerable controversy about the selective forces leading to cooperativity, what are the costs and benefits of cooperativity, and what are the possible advantages to controlling cooperativity by quorum sensing. We will use molecular genetic approaches to address the costs and benefits of controlling cooperative behavior by quorum sensing. Specifically, we aim to study constructed and evolved P. aeruginosa PA01 quorum-sensing mutants and recombinant E. coli to probe the costs and benefits of quorum sensing components and the networks of P. aeruginosa quorum sensing gene control circuits. We will also study naturally occurring quorum sensing mutants from cystic fibrosis patients and use these isolates to understand quorum sensing ?wiring? options and the evolution of quorum sensing signal specificity in P. aeruginosa. By understanding quorum sensing in laboratory experiments and coupling this information to the quorum-sensing circuitry in clinical isolates we can understand how to manipulate quorum sensing and make inferences about conditions prevalent in P. aeruginosa infections. Such information is critical in attempts to develop quorum-sensing technologies for disease control.